Analysis of structure-function relationships on smooth muscle myosin using NMR and X-ray crystallography.

使用 NMR 和 X 射线晶体学分析平滑肌肌球蛋白的结构-功能关系。

• 批准号: 09480172
• 负责人: TANOKURA Masaru
• 金额: $ 8.38万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-09480172/
• 关键词: myosin; smooth muscle; motor protein; NMR; nuclear magnetic resonance; モータータンパク質

Myosin is an enzyme transducing the chemical energy from the hydrolysis of ATP into directed mechanical movement, in conjunction with actin. In this research, we use the NMR, which is a good probe to observe the microenvironments, and X-ray crystallography, which allows us a precise three-dimensional structure determination, to investigate the mechanism of energy conversion by myosin with atomic level.From the pH- and temperature-dependent changes of the signals in the ^<31>P-NMR spectra of smooth muscle sub fragment 1-ADP complex, it is appeared that the interaction between the smooth muscle myosin subfragment 1 and ADP is stronger than that of skeletal muscle myosin subfragment 1 and ADP. In the case of kinesin and ncd motor domains, it was found to exist two different forms of motor domain-ADP complex. In addition, we found a difference between kinesin and ncd motor domains in the chemical shift of the a-phosphate of bound ADP, indicating that the electrostatic or magnetic microenvironments of this site of these motor domains differed from each other.We established the baculovirus expression system to obtain the recombinant chicken gizzard smooth muscle myosin motor domain as well as mutants for crystallization. We also found that the role of the neck region on the ATPase activity of kinesin.

肌球蛋白是一种酶，它与肌动蛋白一起将ATP水解产生的化学能转化为定向机械运动。本研究利用核磁共振技术和X射线晶体学技术，从原子水平上探讨了肌球蛋白的能量转换机制，从<31>平滑肌亚片段1-ADP复合物~ 1 P-NMR谱信号随pH和温度的变化，平滑肌肌球蛋白亚片段1与ADP的相互作用强于骨骼肌肌球蛋白亚片段1与ADP的相互作用。在驱动蛋白和ncd运动域的情况下，发现存在两种不同形式的运动域-ADP复合物。另外，我们还发现驱动蛋白和ncd马达结构域结合ADP的α-磷酸的化学位移存在差异，表明这两种马达结构域结合ADP的α-磷酸的静电或磁微环境不同。我们建立了杆状病毒表达系统，获得了重组鸡肌球蛋白马达结构域及其结晶突变体。我们还发现颈部区域对驱动蛋白ATP酶活性的影响。

项目成果

Ichikawa,T.ラ: "Crystallization and preliminary crystallographic analysis of the sarcosine oxidase from Bacillus sp.NS-129." J.Struct.Biol.120. 109-111 (1997)

Ichikawa, T. La：“芽孢杆菌 NS-129 肌氨酸氧化酶的结晶和初步晶体学分析。J.Struct.Biol.120 (1997)。

Iwasaki, W., Sasaki, H., Nakamura, A., Kohama, K., Tanokura, M.: "Crystallization and preliminary X-ray diffraction studies of a 4OkDa calcium binding protein specifically expressed in plasmodia of Physarum polycephalum"J. Biochem.. 126・1. 7-9 (1999)

Iwasaki, W.、Sasaki, H.、Nakamura, A.、Kohama, K.、Tanokura, M.：“在多头绒泡菌疟原虫中特异性表达的 4OkDa 钙结合蛋白的结晶和初步 X 射线衍射研究”J。生物化学.. 126・1.

Suzuki, Y., Ohkura, R., Sugiura, S., Yasuda, R., Kinoshita, K., Tanokura, M. and Sutoh, K.: "Modulation of actin filament sliding by mutations of the SH2 cysteine in Dictyostelium myosin II."Biochem. Biophys. Res. Commun.. 234(3). 701-706 (1997)

Suzuki, Y.、Ohkura, R.、Sugiura, S.、Yasuda, R.、Kinoshita, K.、Tanokura, M. 和 Sutoh, K.：“网网柄菌肌球蛋白中 SH2 半胱氨酸突变对肌动蛋白丝滑动的调节

Suzuki, Y., Shimizu, T., Morii, H. and Tanokura, M.: "Hydrolysisi of AMPPNP by the motor domain of ncd,a kinesin-related protein."FEBS Lett.. 409(1). 29-32 (1997)

Suzuki, Y.、Shimizu, T.、Morii, H. 和 Tanokura, M.：“NCD（一种驱动蛋白相关蛋白）的运动结构域对 AMPPNP 的水解。”FEBS Lett.. 409(1)。

Suzuki,Y.ラ: "Modulation of actin filament sliding by mutations of the SH2 cysteine in Dictyostelium myosin II." Biochem.Biophys.Res.Commun.234・3. 701-706 (1997)

Suzuki, Y. La：“网网柄菌肌球蛋白 II 中 SH2 半胱氨酸突变对肌动蛋白丝滑动的调节。Biochem.Biophys.Res.Commun.234·3 (1997)”